The invention relates to detection of Enterobacteriaceae and identification of the genus or genera of Enterobacteriaceae present in a test sample. In particular, the invention provides methods for detecting both nucleic acids and the encoded enzyme (deoxyguanosine triphosphate triphosphohydrolase) found only in Enterobacteriaceae. The nucleic acids and methods provided by the invention are useful for determining whether food, water and other types of samples are contaminated with enteric bacteria. Methods of the invention also permit identification of the type of enteric bacteria present in a contaminated sample.
Food poisoning and other food borne diseases that are caused by enteropathogenic bacteria account for millions of illnesses and thousands of deaths each year in the United States (1,2). The clinical conditions that result from acute ingestion of pathogenic bacteria include diarrhea, vomiting, and dysentery (3). However, other more serious medical complications may occur, such as renal and cardiac disorders, neurological dysfunction, hemolytic uremia, and death (4). The situation in non-industrialized countries is even worse, where it is estimated that more than 10 percent of the population is chronically inflicted with food borne disease (5). Public health organizations have not only been faced with an ever increasing rate of food poisoning cases in the United States, but with newly emerging bacterial food borne diseases (6, 7). In addition to human health issues, food borne illnesses take a continued and a heavy economic toll on society by lowering economic productivity and by stretching the available resources of local and national public health organizations (8).
The bacteria responsible for these human illnesses are from the taxonomic family Enterobacteriaceae (9). The four main genera of bacteria within this family that pose a risk to human health via food borne illnesses are: Escherichia, Salmonella, Shigella, and Yersinia. All foodstuffs are susceptible to bacterial contamination of these bacteria. The original sources of this contamination may be from animal hosts (for example, cows, chickens, or pigs) that harbor systemic infections, from improper handling of otherwise uncontaminated foodstuffs (for example, poor worker hygiene), or from washing foodstuffs in contaminated water.
Traditional food and restaurant inspection techniques have relied upon visual inspection of foodstuffs and food preparation areas. However, foodstuffs contaminated with enteropathogenic bacteria often look, smell and taste normal. Many of these pathogens may also survive the cooking process (10, 11). When bacterial culturing is conducted, samples must be returned to a laboratory for microbiological testing. Such tests often take weeks to perform. Meanwhile, a potential health risk continues.
Studies by Quirk and Bessman (12) have revealed that a dGTPase is only detected in bacteria belonging to the family Enterobacteriaceae. However, this reference does not provide nucleic acids probes and antibodies capable of detecting Enterobacteriaceae in general and also distinguishing between the various types of Enterobacteriaceae.
It is therefore imperative to develop faster and more reliable detection methods that are sensitive and specific enough to identify not only that enteropathogenic bacterial contamination exists in food and water samples, but what type of enteropathogenic bacterial contamination is present.
According to the invention, the enzyme deoxyguanosine triphosphate triphosphohydrolase (dGTPase; E.C. 3.1.5.1) is found only in Enterobacteriaceae and detection of this enzyme is a specific indicator that Enterobacteriaceae pathogens are present in a test sample. The invention provides methods for identifying which genus or genera of Enterobacteriaceae is present in a contaminated sample, as well as antibodies and nucleic acids useful for detection of Enterobacteriaceae dGTPase. Such methods may involve immunological, enzymatic, hybridization, nucleic acid amplification and related procedures for detection and identification of Enterobacteriaceae.
The invention provides an isolated nucleic acid that includes SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. Such nucleic acids can selectively hybridize to DNA from a bacteria of the family Enterobacteriaceae.
The invention also provides an isolated nucleic acid that includes SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. These nucleic acids can selectively hybridize to DNA from Escherichia coli even in the presence of DNA from at least one other bacterial species of the family Enterobacteriaceae such as Klebsiella, Salmonella, Shigella or Yersinia.
The invention further provides an isolated nucleic acid that includes SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. These nucleic acids can selectively hybridize to DNA from Salmonella typhymurium, in the presence of DNA from Klebsiella or Escherichia.
The invention also provides an isolated nucleic acid that includes SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. These nucleic acids can selectively hybridize to DNA from Klebsiella oxytoca, in the presence of DNA from Salmonella or Escherichia.
The invention further provides a biosensor chip that includes a solid support and a nucleic acid including SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.
The invention also provides a method of detecting the presence of enteric bacteria in a test sample that includes contacting the test sample with probe under stringent hybridizations conditions, and detecting hybridization between the probe and a nucleic acid in the test sample. Such a probe can include a nucleic acid that includes, for example, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. Such enteric bacteria are of the family Enterobacteriaceae. This method may further include DNA amplification, for example, polymerase chain reaction.
The invention further provides a method of detecting the presence of any species of enteric bacteria in a test sample that includes contacting the test sample with probe under stringent hybridizations conditions, and detecting hybridization between the probe and a nucleic acid in the test sample. Probes useful in this method include nucleic acids with SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. This method may further include DNA amplification, for example, polymerase chain reaction.
The invention also provides a method of detecting the presence of Escherichia in a test sample that includes contacting the test sample with probe under stringent hybridizations conditions, and detecting hybridization between the probe and a nucleic acid in the test sample. Such a probe may be an isolated nucleic acid that includes SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. These probes can selectively hybridize to DNA from Escherichia coli in the presence of DNA from Klebsiella, Salmonella, Shigella or Yersinia. This method may further include DNA amplification, for example, polymerase chain reaction.
The invention further provides a method of detecting the presence of Salmonella in a test sample that includes contacting the test sample with probe under stringent hybridizations conditions, and detecting hybridization between the probe and a nucleic acid in the test sample. Such a probe may be an isolated nucleic acid that includes SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO: 14. These probes can selectively hybridize to DNA from Salmonella typhymurium in the presence of DNA from Klebsiella or Escherichia. This method may further include DNA amplification, for example, polymerase chain reaction.
The invention also provides a method of detecting the presence of Klebsiella in a test sample that includes contacting the test sample with probe under stringent hybridizations conditions, and detecting hybridization between the probe and a nucleic acid in the test sample. Such a probe may be an isolated nucleic acid that includes SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. These probes can selectively hybridize to DNA from Klebsiella oxytoca in the presence of DNA from Salmonella or Escherichia. This method may further include DNA amplification, for example, polymerase chain reaction.
The invention also provides method for detecting enteric bacteria in a test sample that includes contacting a test sample with a biosensor chip that has a solid support and an antibody that can bind to dGTPase from Enterobacteriaceae, and detecting whether dGTPase is bound to the biosensor chip. Biosensor chips that include such a solid support and an antibody that can selectively bind to dGTPase from Enterobacteriaceae are also provided by the invention.
Antibodies that can bind to dGTPase from Enterobacteriaceae, include, for example, antibodies directed against a peptide having SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.